Nov 22, 2025

Modified ZymoBIOMICS™ DNA/RNA Miniprep Kit for parallel extraction of DNA and RNA from peat soil

 Forked from Modified ZymoBIOMICS™ DNA/RNA Miniprep Kit for parallel extraction of DNA and RNA from peat soil
DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpmnk8gzp/v1
  • 1Lund University
Rhiannon Mondav
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpmnk8gzp/v1
External link: https://files.zymoresearch.com/protocols/_r2002_zymobiomics_dna-rna_miniprep_kit.pdf
Protocol Citation: Rhiannon Mondav 2025. Modified ZymoBIOMICS™ DNA/RNA Miniprep Kit for parallel extraction of DNA and RNA from peat soil. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpmnk8gzp/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 22, 2025
Last Modified: November 22, 2025
Protocol  Integer ID: 233161
Keywords: soil, DNA RNA co-extraction, Zymo miniprep, rna standard zymo dna, rna miniprep kit for parallel extraction, rna miniprep extraction protocol, rna miniprep kit, rna, parallel extraction, dna, clogged filters, filter overload, peat, organic soil, modified zymo dna, rna from peat soil, zymo dna, peat soil, peat, filter with standard protocol reduced sample mass, clogged filter, standard protocol reduced sample mass, organic soil
Funders Acknowledgements:
Biodiversity and Ecosystem services in a Changing Climate
Grant ID: SEED grant 2024
Abstract
Modified ZYMO DNA/RNA miniprep extraction protocol written in a user friendly way to reduce in-process-decisions and process time. Sample type is organic soil, peat, from a degraded rich fen

This protocol includes two modifications to tackle clogged filters.

  • Samples were centrifuged for 45 seconds instead of 30 seconds in first pass through filters as 100 ul to 200 ul sample remained trapped above filter with standard protocol
  • Reduced sample mass to reduce clogging of columns and filters, trialled 200 mg, 150 mg, 100 mg compared to 250 mg
Guidelines
Follow standard precautions for working with environmental samples and RNA i.e. clean/sterilise with 70% ethanol and 10 to 30 min UV if available, further remove DNA and RNases from work environment and equipment using a cleaner of your choice. Work in a designated RNA/pre-PCR area or cabinet. Wear a face mask if a risk of sneezing or coughing exists and you don't have a cabinet. Wear latex or nitrile gloves and change often.
Materials
ZymoBIOMICS™ DNA/RNA Miniprep KitZymo ResearchCatalog #R2002


KIT PROVIDED:
solutions and buffers:
DNA/RNA ShieldZymo ResearchCatalog #R1100-50 DNA/RNA Lysis BufferZymo ResearchCatalog #D7001-1-50
DNA/RNA Prep bufferZymo ResearchCatalog #D7010-2-50
DNA/RNA Wash Buffer (concentrate)Zymo ResearchCatalog #D7010-3-24
ZymoBIOMICS™ DNase/RNase-Free WaterZymo ResearchCatalog #D4302-5-50
ZymoBIOMICS™ HRC Prep SolutionZymo ResearchCatalog #D7010-2-50

enzymes:
DNase I SetZymo ResearchCatalog #E1010 or #E1009-A
both ##SKU have 250U DNase I and come with 4 ml DNA Digestion Buffer

tubes, filters, columns:
ZR BashingBead™ Lysis Tubes (0.1 & 0.5 mm)Zymo ResearchCatalog #S6012-50
Spin-Away Filter (yellow)Zymo ResearchCatalog #C1006-50-F
Zymo-Spin™ IIICG Column (green)Zymo ResearchCatalog #C1006-50-G
Zymo-Spin™ III-HRC FilterZymo ResearchCatalog #C1058-50
Collection TubesZymo ResearchCatalog #C1001-500



USER PROVIDED:
consumables:
- pipette tips
- Ethanol 70 % for cleaning
- sharpie
- freezer boxes

Fisherbrand™ Nitrile Indigo Disposable Gloves PPE Cat IIIThermo FisherCatalog #17182182
Kimberly-Clark™ Kimtech™ Science Precision WipesThermo ScientificCatalog #10180601 Thermo Scientific™ Snap Cap Low Retention Microcentrifuge TubesThermo FisherCatalog #11569914 Thermo Scientific™ Snap Cap Low Retention Microcentrifuge TubesThermo ScientificCatalog #11579914
Invitrogen™ Nuclease-Free Water (not DEPC-Treated) Invitrogen - Thermo FisherCatalog #AM9932
Ethanol, Absolute (200 Proof), Molecular Biology Grade, Fisher BioReagents™Fisher ScientificCatalog #16606002
Thermo Scientific™ RNase AWAY™ Surface DecontaminantThermo Fisher ScientificCatalog #14-754-34


equipment:
- mass balance for weighing tubes/samples
- Vortex
- Micro-centrifuge
- Freezers -80 °C
- Freezer -20° C
- optional choice between Nanodrop, gel electrophoresis, Qubit, TapeStation, and/or BioAnalyzer to characterise quantity, purity, and quality of extracted nucleic acids
Equipment
NanoDrop™ 2000
NAME
Microvolume Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-2000
SKU
https://www.thermofisher.com/order/catalog/product/ND-2000
LINK

Equipment
2100 Bioanalyzer
NAME
Fragment Analysis system
TYPE
Agilent
BRAND
G2939BA
SKU
https://www.agilent.com/en/product/automated-electrophoresis/bioanalyzer-systems/bioanalyzer-instrument/2100-bioanalyzer-instrument-228250
LINK

Equipment
FinnPipette
NAME
F2
TYPE
ThermoScientific
BRAND

Equipment
MP Bio FastPrep 24
NAME
bead beating grinder
TYPE
MP Bio
BRAND
116004500
SKU
https://www.mpbio.com/us/fastprep-24-classic-instrument-1-each
LINK



Before start
  • Before starting you will need to clean and de-contaminate work space and equipment where possible

  • Add ethanol to DNA/RNA Wash Buffer  (step 1.2) (once per kit)
  • Reconstitute DNase and aliquot (step 1.3), freeze unused aliquots at - 20°C (once per kit)
  • Aliquot provided water into 1.5 ml or 2 ml tubes
  • Cut pipette tip ends off from one box of 1000 ul tips to transfer slurry (step 2)

  • Perform all steps at room temperature unless specified
Pre-prep
46m

Note: if you have a micro-centrifuge with 24 places, you can process maximum 12 samples per batch because steps 10 to 16 process DNA and RNA simultaneously, ie 12 samples x 2 (DNA + RNA) = 24
20m
  • If samples are frozen at -80°C in DNA/RNA Shield, retrieve now and place on ice to thaw during preparation (see step 2). Remember to check and vortex periodically during set-up.
  • Retrieve DNase from freezer if already reconstituted and place on ice to thaw
(If not already done) Add 96 ml 100% ethanol (104 ml 95% ethanol) to the 24 ml DNA/RNA Wash Buffer concentrate 
  • this only needs to be done once per kit
3m
(If not already done) Reconstitute lyophilized DNase I with DNase/RNase-Free Water, mix by gentle inversion and store frozen aliquots @ -20°C
  • this only needs to be done once per kit
2m
Prepare DNase Master Mix and store on ice until needed:

ABCDEF
sample no.1681224
DNase I (ul)5304060120
DNase dgstn buffer (ul)754506009001800

5m
Pipette 400 µl of DNA/RNA Lysis Buffer into one nuclease-free tube (not provided) for each sample to be processed (see step 4). Label with sample ID
3m
Pipette 800 µl of 95-100% ethanol into one nuclease-free tube (not provided) for each sample to be processed (see step 7). Make sure cap is tightly closed. Label with sample ID and 'RNA'
3m
for each sample:
  • Prepare and label a further 4 tubes with sample ID including 2 with 'RNA' and 2 with 'DNA'.
  • Set up and label 5 collection tubes with sample ID including 2 with 'DNA', and 2 with 'RNA'
  • Place a yellow filter in one collection tube labelled DNA
  • Place a green column in one collection tube labelled RNA
  • Place HRC filters in two collection tubes with one each labelled DNA and RNA
10m
SAMPLE LYSIS
55m
Sample thaw and transfer protocol:
  • For liquid nitrogen flash frozen samples transfer 0.20, 0.15, 0.10 g sample to a ZR BashingBead Lysis Tube and add 750 µl DNA/RNA Shield to lysis tube, cap tightly and vortex briefly, also vortex briefly during thaw 2 or 3 more times. (measure and record mass of sample)
OR
  • For samples collected in DNA/RNA Shield, vortex briefly every five minutes while thawing on ice (may take 30 minutes or longer). Set pipette to 50 µl more than chosen volume, vortex sample briefly or invert to mix, and transfer 900 ul, 800 ul, or 750 µl of slurry (using a pre-cut pipette tip) to a ZR BashingBead Lysis Tube and cap tightly.
10m
  1. Label side of BB Lysis Tube with sample ID as lid labels wear off during bead beating
  2. Bead beat on MP Bio FastPrep for 60 seconds at 6.5 m/s, then
  3. rest for 5 minutes.
  4. Repeat four more times for a total of 5 minutes bead beating
40m
  1. Centrifuge samples 16 000 x g for 30 seconds and
  2. transfer 400 µl of the supernatant to pre-prepared tube with lysis buffer (see step 1.5), and
  3. mix well by inversion. (sample volume is now 800 ul)
5m
DNA
8m
  1. Pour or pipette the sample into a Spin-Away™ Filter (yellow) in a collection tube and
  2. centrifuge 16 000 x g for 45 seconds.
5m
Transfer the Spin-Away™ Filter (yellow) containing DNA into a new Collection Tube and store until ready to proceed with step 10. **SAVE the flow through for step 7!
3m
RNA
35m
  1. Transfer flow-through (800 ul) from step 6 to pre-prepared tube with same volume of ethanol, close cap tightly and mix well by inversion. This contains RNA, sample volume is now 1600 ul.
5m
  1. Transfer 700 ul of the RNA mixture into a prepared Zymo-Spin™ IIICG Column (green) in a Collection Tube
  2. centrifuge 16 000 x g for 45 seconds.
  3. Discard flow through.
  4. Repeat steps 8.1 to 8.3 until all RNA solution has passed through the green column.
10m
  1. Add 400 µl DNA/RNA Wash Buffer to each green column
  2. centrifuge 16 000 x g for 45 seconds
  3. discard the flow-through
  4. Add 80 ul DNase I Master Mix (step 1.4) to green column
  5. incubate @ room temp for 15 minutes
20m
Parallel DNA and RNA processing
43m
  1. Add 400 µl DNA/RNA Prep Buffer to the yellow filter / green column
  2. centrifuge 16 000 x g for 30 seconds
  3. Discard the flow-through.
5m
  1. Add 700 µl DNA/RNA Wash Buffer to the yellow filter / green column
  2. centrifuge 16 000 x g for 30 seconds
  3. Discard the flow-through.
5m
  1. Add 400 µl DNA/RNA Wash Buffer to the yellow filter / green column
  2. centrifuge for 2 minutes 16 000 x g to ensure complete removal of the wash buffer
  3. Carefully transfer the column into a tube (not provided)
  4. discard tube and flow-through
5m
  1. Add 100 µl ZymoBIOMICS™ DNase/RNase-Free Water directly to the yellow filter / green column matrix
  2. incubate for 5 minutes
  3. centrifuge 16 000 x g for 30 seconds to elute DNA and RNA from the respective yellow filter / green column
  4. You can pause here (store in fridge, on ice, or freeze), but make sure sample is thawed and RT before proceeding
8m
  1. add 600 µl ZymoBIOMICS™ HRC Prep Solution to a Zymo-Spin™ III-HRC Filter in a Collection Tube
  2. Centrifuge at 8,000 x g for 3 minutes, discard collection tube and flow through
  3. Transfer prepared III-HRC Filter to a new tube (not provided)
5m
  1. Transfer the eluted DNA and RNA (step 13) into a prepared Zymo-Spin™ III-HRC Filter
  2. centrifuge at exactly 16,000 x g for 3 minutes
5m
  1. Discard filter, close tube lid, place on ice
  2. Pipette 3 to 10 ul of each sample to new tubes for purity, quantity, and quality testing
  3. Place actual samples in -20°C or -80°C freezers for storage.
  4. Optionally make 5 ul and 10 ul aliquots for downstream testing and processing to reduce contamination and degradation risk.

10m